This study established a high-efficiency fluorescence quenching approach for the in situ visualization and modeling of the spatial distribution of xylanase, β-glucosidase, and phosphatase activities in plant leaves under pollution stress (namely, the leaf zymography technique, LZT). In the LZT, a membrane saturated with an enzyme-specific fluorescent substrate on the leaf surface was incubated and the fluorescence image generated on the membrane under ultraviolet light was recorded. An image-based modeling method for restoring the morphological traits of the true image by reducing noise was developed to ensure the accurate estimation of enzyme activities. The LZT could simultaneously measure 48 samples within 2 h, with good reproducibility. The results obtained by the LZT were comparable to those obtained by a conventional biochemical analysis method and presented lowcost and convenient advantages. This paper explains the theoretical basis required to investigate the realistic application of the LZT for assessing ecotoxicity in large-scale monitoring.